Zinc formal fixx

Freshly dissected tissues were rinsed with PBS and fixed with 1× Zinc Formal-Fixx (Thermo Fisher Scientific). Fixed tissues were then dehydrated with ethanol and embedded in paraffin (Surgipath Paraplast Plus, Leica Biosystems Division of Leica, Buffalo Grove, IL, USA). Blocks were sliced into 4 μm sections and stained with H&E (hematoxylin and eosin) and alcian blue solution (pH 2.5).

HUAFs were cultured on coverslips and transfected with GM-ctrl or GM-113-6 for 24 h. For collagen staining, cells were fixed in Zinc Formal-Fixx (Thermo Fisher Scientific, cat. no. 6764255) for 30 min and washed with PBS. Collagen was visualized with a picrosirius red staining.
For αSMA staining, cells were fixed in 4% PFA for 15 min and washed in PBS. An antibody against αSMA, 1A4 (Dako M0851, 1:1000), and a secondary antibody Alexa Fluor 555 DαMouse (Invitrogen A31570, 1:1000) were used to visualize αSMA. Hoechst (34580, 1:1,000) was used to stain the nuclei.
Fiji was used to perform immunohistochemistry and immunofluorescence analysis. The area was divided by the total amount of nuclei. The integrated density, which is the sum of values of the pixels, was calculated and divided by the total amount of nuclei (intensity).
Primer sequences for COL1A1 and αSMA (smooth muscle α-2 actin [ACTA2]) are provided in Table S1.

Mouse liver samples were fixed in zinc formal‐fixx (ThermoFisher Scientific) and embedded in paraffin. Hematoxylin (ThermoFisher Scientific) and eosin (ThermoFisher Scientific; H&E) stained slides were used for histology evaluation. Immunohistochemistry was performed as described previously.13 In brief, slides were deparaffinized in xylene, rehydrated through a graded alcohol series and finally, rinsed in PBS. Antigen unmasking was performed via boiling the slides in 0.01 mol/L citrate buffer (pH 6.0) for 10 minutes. The slides were first incubated with 5% goat serum and Avidin‐Biotin Blocking Kit (Vector Laboratories, Burlingame, CA, USA); and, subsequently, with primary antibodies at 4°C for 16 hours. After washing, slides were incubated with 3% hydrogen peroxide for 10 minutes followed by incubating with biotin conjugated secondary antibody for additional 30 minutes at room temperature. Vectastain ABC Elite Kit (Vector Laboratories) was used to visualize the signal. Finally, slides were counterstained with Hematoxylin. Slides incubated without primary antibodies were used as negative control. Images were taken using bright‐field microscope with Digital color camera (Model# DFC295; Leica, San Francisco, CA, USA). Detailed information for the reagents could be found in Table S1 and antibody information in Table S2.